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- EMDB-13856: Tomogram of respiratory syncytial virus filamentous virion -

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Basic information

Entry
Database: EMDB / ID: EMD-13856
TitleTomogram of respiratory syncytial virus filamentous virion
Map dataRepresentative tomogram of filamentous respiratory syncytial virus
Sample
  • Virus: Respiratory syncytial virus A2
    • Organelle or cellular component: Budding RSV Virion
KeywordsVirion / VIRUS
Biological speciesRespiratory syncytial virus A2
Methodelectron tomography / cryo EM
AuthorsConley MJ / Vijayakrishnan S / Bhella D
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UU_12014/7 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/M000451/1 United Kingdom
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI113321 United Kingdom
Wellcome Trust102463/Z/13/Z United Kingdom
Wellcome Trust099786/Z/12/Z United Kingdom
CitationJournal: EMBO J / Year: 2022
Title: Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.
Authors: Michaela J Conley / Judith M Short / Andrew M Burns / James Streetley / Joshua Hutchings / Saskia E Bakker / B Joanne Power / Hussain Jaffery / Joanne Haney / Giulia Zanetti / Pablo R Murcia ...Authors: Michaela J Conley / Judith M Short / Andrew M Burns / James Streetley / Joshua Hutchings / Saskia E Bakker / B Joanne Power / Hussain Jaffery / Joanne Haney / Giulia Zanetti / Pablo R Murcia / Murray Stewart / Rachel Fearns / Swetha Vijayakrishnan / David Bhella /
Abstract: Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image ...Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.
History
DepositionNov 5, 2021-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13856.png

Downloads & links

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Map

FileDownload / File: emd_13856.map.gz / Format: CCP4 / Size: 56 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRepresentative tomogram of filamentous respiratory syncytial virus
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.79 Å/pix.
x 3710 pix.
= 6640.9 Å		1.79 Å/pix.
x 1056 pix.
= 1890.24 Å		1.79 Å/pix.
x 3838 pix.
= 6870.02 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.79 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-169.063520000000011 - 162.010559999999998
Average (Standard dev.)0.0028832706 (±22.863993000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions105638383710
Spacing383810563710
CellA: 6870.02 Å / B: 1890.24 Å / C: 6640.9 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Respiratory syncytial virus A2

EntireName: Respiratory syncytial virus A2
Components
  • Virus: Respiratory syncytial virus A2
    • Organelle or cellular component: Budding RSV Virion

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Supramolecule #1: Respiratory syncytial virus A2

SupramoleculeName: Respiratory syncytial virus A2 / type: virus / ID: 1 / Parent: 0
Details: Sub tomogram averaging was performed focusing on the envelope of filamentous virions propagated directly on the cryo-EM grid.
Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Budding RSV Virion

SupramoleculeName: Budding RSV Virion / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Respiratory syncytial virus A2

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/2 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
Details: Grids were sterilised in EtOH and pretreated with laminin.
VitrificationCryogen name: ETHANE
DetailsVirions were propagated in A549 cells, grown on gold quantifoil cryo-EM grids that had been pre-treated with laminin.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI / Diameter: 5 nm

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 2.0 e/Å2
Details: Data collected at the UK electron bioimaging centre at Diamond Light Source (eBIC) on a Thermo-Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered ...Details: Data collected at the UK electron bioimaging centre at Diamond Light Source (eBIC) on a Thermo-Fisher Scientific Titan Krios microscope equipped with a Gatan BioQuantum K2 energy filtered direct detection camera. Dose-symmetric tilt-series collection was performed using SerialEM. Energy-filtered images were collected with a slit-width of 20eV and an applied defocus of between -2 and -4.5 microns. One second exposures were recorded with an electron exposure of 2 electrons/A2, partitioned over five movie frames. A total of 41 images were recorded per tilt-series at 3 degree intervals from -60 to +60, thus a total exposure of 82 electrons/A2 was applied per tilt-series. Tilt-series were recorded at a nominal column magnification of 81k corresponding to a calibrated pixel size of 1.79 A at the specimen scale.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsIndividual movies were corrected for drift and beam induced motion using MotionCor2. Motion corrected images were then compiled into an angle ordered stack file using a perl script to extract the correct files and tilt angles from the SerialEM metadata files (mdoc), creating the final tilt-series using the IMOD command new-stack. Tilt-series alignment and reconstruction by weighted back projection was then accomplished using the IMOD package
Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 41

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